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研究生:陳玉芬
研究生(外文):Chen,YuFen
論文名稱:探討當歸藥渣抑制脂多醣誘導小鼠巨噬細胞 RAW264.7 之發炎反應
論文名稱(外文):The Study Of Angelica Sinensis Residues Suppressing LPS-induced Inflammation In Murine Macrophage Cell Line RAW264.7
指導教授:許惠貞許惠貞引用關係
指導教授(外文):Hsu HuiChen
口試委員:蔡孟峯鄭永祥陳威戎
口試委員(外文):Tsai, MengFengJeng,Yung HsiangChen,WeiJung
口試日期:2012-07-05
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:動物科技學系碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:83
中文關鍵詞:當歸
外文關鍵詞:Angelica sinensis
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中草藥中當歸已被證實具有抗發炎的效用,但尚未有研究針對蒸煮後之當歸藥渣是否具有活性做進一步分析。本研究先對當歸藥渣萃取物活性進行分析,再利用細胞模式進行免疫活性分析,探討當歸藥渣是否仍具抗發炎之功效,以當歸藥渣餵飼乳羊的可行性評估。利用高效液相層析分析當歸指標成份分析水溶性之阿魏酸 (Ferulic Acid) 與脂溶性之蒿本內酯 (Z-ligustilide) 的含量,結果顯示藥廠與市售當歸藥渣與青貯的藥渣中,阿魏酸含量皆隨著蒸煮與青貯後減少,而蒿本內酯在藥渣中含量增加,但青貯過後則是含量減少。此外,利用小鼠巨噬細胞 RAW264.7 與小鼠纖維母細胞 NIH-3T3以評估藥渣與青貯的藥渣萃取物是否具有細胞毒性,結果顯示當歸藥渣對此二細胞均不具細胞毒殺作用,僅於市售當歸仿照藥廠蒸煮方式之藥渣在青貯後的高劑量具有 65% 的細胞毒性。以一氧化氮產量檢測藥廠與市售當歸原料、藥渣與青貯藥渣萃取物之抗發炎功效,結果顯示均可抑制發炎反應產生,且具有劑量依賴性。為了探討藥廠當歸原料、藥渣與青貯的藥渣萃取物之抗發炎機轉,利用西方墨點法分析結果顯示藥渣與青貯藥渣萃取物可減少促發炎因子 IL-1β 、TNF-α 與發炎酵素 COX-2 (Cyclooxygenase-2) 之蛋白質表現,進一步探討細胞核內 NFκB (Nuclear Factor-KappaB) 蛋白質的變化,顯示藥廠當歸原料與藥渣萃取物細胞核內 NFκB 的表現會隨著原料與藥渣劑量增加而減少。綜合以上結果顯示當歸藥渣與藥渣青貯仍殘留指標成分,且具有抗發炎之功效,但青貯藥渣萃取物真正影響抗發炎之成分與機制,仍需要進一步探討,本論文有助於當歸藥渣應用於反芻動物的餵飼評估。
Chinese herb medicine has been used widely during the recent years. The Chinese herb residues which usually have not been utilized except for compost. The chinese herb residues contain mainly plant fiber which could be used to asa feed for animals. Angelica sinensis, chinese herb medicine, is known to have anti-inflammatory effects. However, there is no any report respect to the biofuntions of Angelica sinensis residues and Angelica sinensis residues sliage. The objective initially was to investigate the anti-inflammatory activity of Angelica sinensis residues and Angelica sinensis residues silage in vitro, evaluating the effects of feeding herb residues to dairy goats. Two active components for ferulic acid and Z-ligustilide in Angelica sinensis, residues and silage were dctermined by HPLC. Furthermore, the murine macrophage cell line RAW264.7 stimulated with lipopolysaccharide (LPS) cocultured with methanol extractions of Angelica sinensis residues and Angelica sinensis residues sliage. Our showed two kind of extracts significantly suppress the production of nitric oxide and pro-inflammatory cytokines, interleukin (IL)-1β, TNF-α and COX-2. Angelica sinensis residues not only inhibit LPS-induced phosphorylation of inhitor IκB (plκB) but also activated the inhibitor κB (IκB) kinases, and NF-κB nuclear translocation was blocked. However, the underlying anti-inflammatory mechanisms of A. sinensis residues silage have been elucidated in current study. Future investigation need to be done in anti-inflammatory mechanism of A. sinensis residues silage. This study implies that herb residues showing the potential of improving productivity and immunity in ruminants.
中文摘要…………………………………………………………………Ⅰ
Abstract…………………………………………………………………Ⅱ
謝誌………………………………………………………………………Ⅲ
目錄…………………………………………………………………….…Ⅴ
圖目錄………………………………………………………………….....Ⅸ
表目錄 ………………………………………………………………..….XI
壹、前言……………………………………………………………………1
貳、文獻探討………………………………………………………………2
一、當歸……………………………………………………………2
二、當歸作用機制……….…………………………………….….3
(一) 抗癌……………..……………………………………..4
(二) 抗心血管疾病…………………………………………5
(三) 神經保護作用…………………………………………7
(四) 免疫調節………………………………………………8
(五) 抗發炎…..……….………………………………….....8
三、中草藥之動物應用………………………………….……….9
四、動物發炎簡介……………………………………………….13
五、青貯 (sliage ) ……………….……………………..….….…18
參、材料與方法……….…………………………………………………19
一、當歸藥渣指標成分萃取…………………………….………19
(一) 當歸藥渣製備……...……...………………..…………19
(二) 青貯當歸藥渣製備………………..…………………..20
(三) 當歸藥渣之液相萃取製備……………..……………..20
(四) 當歸藥渣之指標成分分析………..………….……….21
二、細胞培養…………………………..…………………..…….22
三、當歸藥渣對細胞存活率之影響…………………………….25
四、當歸藥渣之抗發炎活性分析……………………………….27
(一) 一氧化氮之含量分析………………..….………….…27
(二)西方墨點法 (Weatern blot) ………………..….…..…...28
肆、實驗結果…………………………………………………………….32
一、藥廠當歸原料、藥渣與青貯藥渣之 HPLC 圖譜分析……32
二、藥廠當歸原料、藥渣與青貯藥渣對細胞存活率之影響.…33
三、藥廠當歸原料、藥渣與青貯藥渣於脂多醣刺激小鼠巨噬細胞RAW264.7 之一氧化氮影響………...…………………33
四、藥廠當歸原料、藥渣與青貯藥渣於脂多醣刺激小鼠巨噬細胞 RAW264.7 之發炎因子影響…...……………….…..…34
五、藥廠當歸原料、藥渣與青貯藥渣於脂多醣刺激小鼠巨噬細胞RAW264.7 細胞質與細胞核之NF-κB 路徑影響…….35
六、市售當歸原料、藥渣與青貯藥渣之 HPLC 圖譜分析……36
七、市售當歸原料、藥渣與青貯藥渣對細胞細胞存活率之影響.............................................................................................38
八、市售當歸原料、藥渣與青貯藥渣於 LPS刺激小鼠巨噬細胞 RAW264.7 之一氧化氮影響.….…...……………..…….…38
伍、討論……………………………………………………………….…40
一、當歸藥渣與青貯之指標成分分析…………………………..40
(一) 阿魏酸含量…..………………..……………..…………40
(二) 蒿本內酯……..…………………………………………42
二、當歸藥渣與青貯藥渣對細胞存活率之影響………………43
三、當歸藥渣與青貯藥渣之抗發炎分析………………………44
陸、結論……………………………………………………………….…47
柒、圖 表…………………………………………………………………48
捌、參考文獻……………………………………………………….……66



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